In the past two blog posts we discussed adeno-associated viruses (AAVs) and their purification. In our final post on AAVs, we are focusing on methods for full capsid separation, the final step in obtaining viral particles of sufficient quality for use as gene therapies.

Once impurities in engineered AAVs have been minimized through an initial chromatography step (discussed last time), full and empty capsids can be separated without the risk of interference or noise. The characteristic that differentiates the two is the negative charge associated with the DNA strand contained within the full capsids. This difference means that separation is reliant on anionic exchange chromatography1,2,3,4,5,6. The success of empty capsid removal from the drug substance is imperfect and requires manual intervention for a more purified drug substance in current manufacturing practices1,2,3,4,5,6. If a sample turns out to be insufficiently cleared of empty capsids or other contaminants, the viable capsids must be discarded. This situation creates the need for efficient capsid polishing procedures.

There are two semi-continuous techniques to prevent contamination of the viral particles by empty capsids in modern high-throughput processes. The first utilizes an analytical ultracentrifuge (AUC) to spin the AAVs down into a pellet1,5,7. This pellet can then be collected and manually portioned to collect a predominantly full portion of the capsid sedimentation. For a more continuous process, an in-line spectrophotometer can be used for identification, but it will require an additional step for apportionment of the empty and full capsids. The elution from the column will transition from the empty capsid peak to the full capsid peak while the elution buffer is pumped through the column2,5,8. By combining the spectrophotometer readings and flow meter information, technicians can direct flow to in-line single-use bags for physical separation of the capsids based on these peaks, which are referred to as fractions. A quality control lab will then determine which fractions can be used and which must be discarded.

Since there can be poor resolution between fractions, manufacturers must discard some of the fractions to meet quality specifications and regulatory requirements9. In the case of blockbuster gene therapies (those products expected to make over $1 billion per year in sales), small improvements in the purification and separation process can result in big savings for the manufacturer. As gene therapies become a larger part of the drug market, the efficient and cost-effective production of AAVs (and other virus vectors) will become crucial. Some gene therapy companies are beginning to adopt ideas from other manufacturing processes, and developing platforms that integrate different parts of AAV preparation together, allowing for more effective end-to-end, large-scale manufacturing of AAVs for gene therapies10,11. While these end-to-end, continuous systems are still in under development, there is encouraging progress in simplifying large-scale AAV production11.

This blog series highlighted some of the challenges and approaches to AAV production. If you find yourself needing support in this area, Kymanox is here to be of service to you and your team! Please reach out to our Business Development Team at to learn more.

  1. Dickerson, R., Argento, C., Pieracci, J., & Bakhshayeshi, M. (2021). Separating empty and full recombinant adeno‐associated virus particles using isocratic anion exchange chromatography. Biotechnology Journal16(1), 2000015.
  2. Li, T., Gao, T., Chen, H., Demianova, Z., Wang, F., Malik, M., … & Mollah, S. (2020). Determination of Full, Partial and Empty Capsid Ratios for Adeno-Associated Virus (AAV) Analysis.
  3. Lock, M., Alvira, M. R., & Wilson, J. M. (2012). Analysis of particle content of recombinant adeno-associated virus serotype 8 vectors by ion-exchange chromatography. Human Gene Therapy, Part B: Methods23(1), 56-64.
  4. Nass, S. A., Mattingly, M. A., Woodcock, D. A., Burnham, B. L., Ardinger, J. A., Osmond, S. E., … & O’Riordan, C. R. (2018). Universal method for the purification of recombinant AAV vectors of differing serotypes. Molecular Therapy-Methods & Clinical Development9, 33-46.
  5. Rieser, R., Koch, J., Faccioli, G., Richter, K., Menzen, T., Biel, M., … & Michalakis, S. (2021). Comparison of different liquid chromatography-based purification strategies for adeno-associated virus vectors. Pharmaceutics13(5), 748.
  6. Wang, C., Mulagapati, S. H. R., Chen, Z., Du, J., Zhao, X., Xi, G., … & Liu, D. (2019). Developing an anion exchange chromatography assay for determining empty and full capsid contents in AAV6. 2. Molecular Therapy-Methods & Clinical Development15, 257-263.
  7. Cole, J. L., Lary, J. W., Moody, T. P., & Laue, T. M. (2008). Analytical ultracentrifugation: sedimentation velocity and sedimentation equilibrium. Methods in cell biology84, 143-179.
  8. “Ion Exchange Chromatography Principles and Methods.” GE Healthcare,
  9. Gonçalves, M. A., Pau, M. G., de Vries, A. A., & Valerio, D. (2001). Generation of a high-capacity hybrid vector: packaging of recombinant adenoassociated virus replicative intermediates in adenovirus capsids overcomes the limited cloning capacity of adenoassociated virus vectors. Virology288(2), 236-246.
  10. “Clearing the hurdles of gene therapy manufacturing.” Pharma Manufacturing,
  11. Zhang, P., Marchand, N., Schofield, M., King, D., & Sargent, B. (2022, PALL Corporation) From Bench to Bedside: A Scalable End-to-End Solution for AAV Production, 1-6.